KR20240015755A - Antibacterial Protein Having Lytic Activity to Streptococci - Google Patents

Abstract

The present invention relates to SBL2200, an antibacterial protein that has specific antibacterial activity against streptococci, and more specifically, has the ability to specifically lyse streptococci and has an amino acid sequence represented by SEQ ID NO: 1. It relates to a streptococcus-specific antibacterial protein SBL2200, and a pharmaceutical composition for treating infections caused by streptococci, comprising the streptococcus-specific antibacterial protein SBL2200 as an active ingredient.

Description

Antibacterial protein having lytic activity against streptococci {Antibacterial Protein Having Lytic Activity to Streptococci}

The present invention relates to SBL2200, an antibacterial protein that has lytic activity against streptococci. More specifically, it has the ability to specifically lyse streptococci that can cause infectious diseases, and is indicated by SEQ ID NO: 1. It relates to a streptococcus-specific antibacterial protein SBL2200, which is characterized by having an amino acid sequence, and a pharmaceutical composition for the treatment of infectious diseases caused by streptococci, comprising the streptococcus-specific antibacterial protein SBL2200 as an active ingredient.

Streptococci are Gram-positive bacteria that grow in pairs or chains. Depending on the degree of hemolysis, they can be either α-hemolytic (partial hemolysis), β-hemolytic (complete hemolysis), or complete hemolysis. ), and γ-hemolytic (No hemolysis; enterococcus), α-hemolytic is further subdivided according to biochemical characteristics (Optochin susceptibility), and β-hemolytic is the serological characteristics of C-polysaccharide produced during metabolism. It is further subdivided into Group (serotype) A~V according to (Lancefield classification).

Streptococci are widely distributed in nature, and some species belong to the normal human flora, but S. pneumoniae , S. pyogenes , and S. agalactiae are known to be serious pathogens. Infections caused by these pathogenic streptococci include bacteremia, endocarditis, abscess, tooth decay, pneumonia, meningitis, sinusitis, otitis media, pharyngitis, scarlet fever, pyodermatosis, cellulitis, necrotizing fasciitis, rheumatic fever, glomerulonephritis, and neonatal infection (meningitis). , pneumonia, bacteremia), urethral infection, amnionitis, endometritis, wound infection, etc.

Specifically, the main causative agent of Group A streptococcal infections is known to be S. pyogenes , and it mainly causes non-invasive infections such as pharyngitis, pyodermatosis, and cellulitis, as well as rheumatic fever, acute glomerulonephritis, sepsis, and other infections through the throat and skin. It is known to cause various invasive infections such as scarlet fever and bacteremia. As of 2018, tens of millions of non-invasive Group A streptococcal infections occurred in the United States, and it is reported that approximately 12,500 to 20,000 people are infected with invasive Group A streptococci each year, and approximately 1,250 to 1,900 of them die. Antibiotics used to treat Group A streptococcal infections include penicillin, ampicillin, erythromycin, clindamycin, and macrolides, including erythromycin, Antibiotic resistance rates for clindamycin and macrolides are increasing by about 20% every year as of 2017.

The main causative agent of Group B streptococcal infection is known to be S. agalactiae . It mainly lives in the intestinal tract or female genital tract, and is infected in newborns during delivery, causing sepsis and meningitis. In adults, it also causes bacteremia, pneumonia, bone and joint infections, etc. It causes skin and soft tissue infections. As of 2016, approximately 31,000 serious Group B streptococcal infections occurred in the United States, of which approximately 1,700 people were reported to have died. Treatment of these Group B streptococcal infections mainly uses penicillin, clindamycin, and erythromycin, but it has been reported that clindamycin and erythromycin show antibiotic resistance of about 40-60% as of 2016.

In order to effectively deal with streptococcal infections that have acquired resistance to existing antibiotics, the development of new antibacterial substances is necessary, and in particular, the development of pharmaceutical agents with new mechanisms of action is very urgent.

Accordingly, as a solution to the problems of existing antibiotic treatment for harmful pathogenic streptococci, the present inventors provided an antibacterial protein SBL2200 that can specifically lyse streptococci, and further contains this as an active ingredient and treats streptococcal infections. The aim is to provide a pharmaceutical composition that can be used for treating streptococcal infections, and furthermore, to provide a method of effectively treating streptococcal infection using the pharmaceutical composition.

Therefore, the purpose of the present invention is to provide an antibacterial protein SBL2200, which has an amino acid sequence represented by SEQ ID NO: 1 and has antibacterial activity capable of specifically lysing streptococci.

Another object of the present invention is to provide a method for efficiently producing the antibacterial protein SBL2200, which has an amino acid sequence represented by SEQ ID NO: 1 and has antibacterial activity capable of specifically lysing the streptococci. In relation to this, Escherichia as a producing strain of the antibacterial protein SBL2200 coli AB-22. The production strain Escherichia coli AB-22 was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on July 12, 2022 (accession number KCTC15033BP).

Another object of the present invention is to provide a pharmaceutical composition for the treatment of streptococcal infection containing the antibacterial protein SBL2200, which can specifically lyse the streptococci, as an active ingredient.

In addition, another object of the present invention is to provide a method of treating streptococcal infection using a pharmaceutical composition containing the antibacterial protein SBL2200 as an active ingredient.

In order to achieve the above objectives, the inventors of the present invention utilize information on various antibacterial proteins known to have antibacterial activity against various bacterial species, and utilize the knowledge and expertise of the present inventors to manufacture various antibacterial protein candidates in the form of recombinant proteins. By examining their lytic activity against streptococci, we selected antibacterial proteins with excellent lytic activity, developed a method to efficiently manufacture them, and finally, could be used to treat streptococcal infections using them as an active ingredient. The present invention was completed by developing a pharmaceutical composition.

Therefore, according to one aspect of the present invention, the present invention provides an amino acid sequence of the antibacterial protein SBL2200, which can specifically lyse streptococci. Specifically, it corresponds to the amino acid sequence of SEQ ID NO: 1. SBL2200, an antibacterial protein that can specifically lyse streptococci, consists of 279 amino acid residues and has a molecular weight of approximately 31.3 kDa.

It is obvious that the amino acid sequence of SEQ ID NO: 1 can be partially modified by those skilled in the art using known techniques. The modifications include partial substitution of the amino acid sequence, partial addition of the amino acid sequence, and partial deletion of the amino acid sequence. However, it is most preferable to apply the amino acid sequence of SEQ ID NO: 1 disclosed in the present invention.

In addition, the present invention provides a production strain Escherichia that can be used to produce the antibacterial protein SBL2200 having the amino acid sequence of SEQ ID NO: 1. coli AB-22. The producing strain Escherichia coli AB-22 is a strain created to produce SBL2200 by transforming E. coli using a plasmid having the nucleic acid sequence (4,907 bp) of SEQ ID NO: 2 produced by the present inventors.

According to another aspect of the present invention, the present invention is effectively used in the treatment of streptococcal infection containing as an active ingredient the antibacterial protein SBL2200, which is characterized by the amino acid sequence of SEQ ID NO: 1 and has a specific lytic activity against streptococci. Provides a pharmaceutical composition that can be used.

The antibacterial protein SBL2200, which is included in the pharmaceutical composition of the present invention and has a specific lytic activity against streptococci characterized by the amino acid sequence of SEQ ID NO: 1 according to the present invention, specifically lyses streptococci as described above. , It is effective in treating various diseases caused by streptococci. Therefore, the pharmaceutical composition of the present invention can be implemented as an antibiotic, disinfectant, disinfectant, therapeutic agent, etc. and used to treat various diseases caused by streptococci.

Therefore, according to another aspect of the present invention, the streptococcus-specific antibacterial protein SBL2200, which is characterized by having an amino acid sequence represented by SEQ ID NO: 1, is administered to an individual with a streptococcal infection to prevent streptococcus infection. Provides methods to treat various diseases caused by it.

In this specification, “disease caused by streptococci” refers to a disease caused by streptococcal infection, including bacteremia, endocarditis, abscess, cavities, pneumonia, meningitis, sinusitis, otitis media, pharyngitis, scarlet fever, pyodermatosis, cellulitis, and necrosis. It refers to, but is not limited to, fasciitis, rheumatic fever, glomerulonephritis, neonatal infections (meningitis, pneumonia, bacteremia), urethral infections, amnionitis, endometritis, and wound infections.

Streptococci in this specification may be sensitive to existing antibiotics or resistant to existing antibiotics. In other words, it does not matter whether resistance to existing antibiotics is acquired or not.

As used herein, the term “treatment” or “treatment” refers to all actions that inhibit infection caused by streptococci and alleviate the pathological condition of diseases caused by streptococci.

In order to increase efficiency for this purpose, antibacterial substances that can provide antibacterial activity against other bacterial species can be added to the pharmaceutical composition of the present invention.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

The pharmaceutical composition of the present invention may be administered through oral or parenteral administration, and in the case of parenteral administration, it may be administered using intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration. It can also be used by applying or spraying to the diseased area, but is not limited to this.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. Alternatively, it may be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer.

In addition, suitable application, spraying and dosage of the pharmaceutical composition will depend on factors such as formulation method, administration mode, age, body weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and reaction sensitivity. It varies depending on the dose, and usually a skilled doctor or veterinarian can easily determine and prescribe an effective dosage for the desired treatment.

The pharmaceutical composition of the present invention can be implemented as an antibiotic, disinfectant, disinfectant, therapeutic agent, etc.

The method of treating streptococcal infection using a pharmaceutical composition containing the antibacterial protein SBL2200 having the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient according to the present invention is effective even against streptococci that have acquired resistance to existing antibiotics or antibacterial substances. It can work. Meanwhile, the streptococcus-specific antibacterial protein SBL2200 of the present invention does not affect normal flora other than streptococci, and can minimize side effects resulting from the use of a pharmaceutical composition containing the antibacterial protein SBL2200 as an active ingredient. Existing antibiotics have the disadvantage of having a negative effect on beneficial bacteria and causing various side effects.

Figure 1 is an electrophoresis photograph showing the results of producing the streptococcus-specific antibacterial protein SBL2200, which is characterized by having the amino acid sequence represented by SEQ ID NO: 1, in the form of a recombinant protein, where lane M is a protein size marker, and lane 1 is 2. It is the chromatographic solution used during tea refining.

Hereinafter, the present invention will be described in more detail based on examples, but these examples are only illustrative of the present invention and the scope of the present invention is not limited to these examples.

Example 1: Streptococcus specific antibacterial protein SBL2200's manufacturing

The production of streptococcus-specific antibacterial protein SBL2200 is described below. In this example, Escherichia, a strain produced by transforming E. coli using a plasmid having the nucleic acid sequence of SEQ ID NO. 2 produced by the present inventors coli AB-22 was used as the production strain.

Escherichia in 20 ml of LB medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) containing 50 μg/ml kanamycin. coli AB-22 was added to inoculate 20 μl and then cultured with shaking at 37°C overnight. The next day, the overnight culture medium was added at a volume ratio of 1/100 to 1 L of LB medium containing 50 μg/ml kanamycin. Cultivation was performed at a temperature of 37°C with a stirring speed of 200 rpm. When the cell concentration reached 0.3 to 0.35 based on absorbance at 600 nm, the culture temperature was changed to 25°C, and about 20 minutes later, when the cell concentration reached 0.5 based on absorbance at 600 nm, the final Expression of the antibacterial protein SBL2200 with the amino acid sequence shown in SEQ ID NO: 1 was induced by adding L-arabinose at a concentration of 0.2%, and then cultured for an additional 6 hours.

After completion of incubation, the cell culture fluid was taken and centrifuged at 6,000 rpm for 10 minutes at 4°C to recover the cell sediment. The recovered cell sediment was suspended in 40 ml of buffer (50 mM Potassium phosphate, 2 M NaCl, pH 7.5). . The cell suspension prepared in this way was disrupted using ultrasonic disruption. The ultrasonic disruption method involves applying ultrasonic waves for 3 seconds to break the cells, stopping for 3 seconds, and repeating this process for a total of 15 minutes in an ice bath. It was carried out in .

After cell disruption, the cell disruption solution was centrifuged at 13,000 rpm for 20 minutes at 4°C, and the obtained supernatant was purified through conventional hydrophobic interaction chromatography and anion-exchange chromatography purification processes. did.

The purification process is briefly explained as follows. In this example, 5 ml of HiTrap ^TM Phenyl HP (GE Healthcare) was used as a hydrophobic interaction resin and 5 ml of HiTrap ^TM Q HP (GE Healthcare) was used as an anion-exchange resin. did. As a first purification process, hydrophobic interaction chromatography was performed after pre-equilibrating the column with Buffer A (50 mM Potassium phosphate, 2 M NaCl, pH 7.5), and after dropping the sample on the column, the flow rate was 5 ml/min. Washing was performed by flowing 5 CV (Column Volume) of buffer A at (flow rate). After washing, hydrophobic interaction chromatography, which is the first purification process, is performed under the condition that the concentration gradient from buffer A to buffer B (50 mM potassium phosphate, pH 7.5) is from 0% to 100% at a flow rate of 5 ml/min. The graph was performed. Next, the second purification process, anion-exchange resin chromatography, was performed using the obtained first purification eluate. Anion-exchange resin chromatography was performed after pre-equilibrating the column with buffer B (50 mM Potassium phosphate, pH 7.5), and the sample obtained in the first purification process was added dropwise to the column and then purified at a flow rate of 5 ml/min. Washing was performed by flowing 10 CV under conditions such that buffer C (50 mM Potassium phosphate, 1 M NaCl, pH 7.5) was 30% in buffer B. After washing, chromatography was performed at a flow rate of 5 ml/min under conditions such that the concentration gradient from buffer B to buffer C was 30% to 100%. In this process, elution of the antibacterial protein SBL2200 with the desired amino acid sequence represented by SEQ ID NO: 1 was achieved. Figure 1 shows the results of electrophoresis analysis of the antibacterial protein SBL2200 purified using a chromatographic purification process.

Among the obtained purified fractions, fractions containing a high concentration of the antibacterial protein SBL2200 were collected, and medium exchange was performed by dialyzing them against a buffer solution (50 mM potassium phosphate, pH 7.5). Through this, it was possible to secure a solution of antibacterial protein SBL2200 with a purity of over 90%.

Buffer exchange was performed on the concentrated SBL2200 solution with buffer (50 mM Potassium phosphate, 5% (w/v) Sorbitol, 0.1% (w/v) poloxamer 188, pH 7.5) to produce 'SBL2200 pharmaceutical composition. ' was manufactured.

Example 2: antibacterial protein SBL2200's Antibacterial activity investigation

The antibacterial activity of the streptococcus-specific antibacterial protein SBL2200 prepared according to Example 1 was investigated. The details of the target strains used to investigate antibacterial activity are listed in Table 1.

Strains subject to antibacterial activity investigation fungus Strain Source Streptococcus agalactiae ATCC 27956 ATCC CCARM 4523 CCARM CCARM 4537 Streptococcus pyogenes CCARM 4528 CCARM CCARM 4533 Staphylococcus aureus ATCC 33591 ATCC CCARM 3090 CCARM Acinetobacter baumannii CCARM 12202 CCARM Clostridioides difficile CCARM 0186 CCARM Pseudomonas aeruginosa ATCC BAA-47 ATCC Klebsiella pneumoniae CCARM 10332 CCARM Escherichia coli CCARM 1G938 CCARM CCARM: Culture Collection of Antimicrobial Resistance Microbes;
ATCC: American Type Culture Collection

Turbidity reduction assay and MIC assay were used to investigate antibacterial activity. The turbidity reduction survey method was conducted as follows. Suspend the bacteria to be tested in physiological saline so that the absorbance is about 0.7 at 600 nm, add 0.1 ml of antibacterial protein SBL2200 solution to 0.9 ml of this suspension (final concentration: 2.5 μM), and measure the absorbance at 600 nm for 30 minutes. It was carried out in this way. As a negative control, a buffer (50 mM Potassium phosphate, 5% (w/v) Sorbitol, 0.1% (w/v) poloxamer 188, pH 7.5) that does not contain the antibacterial protein SBL2200 was used instead of the antibacterial protein SBL2200 solution. MIC was derived by conducting a routine MIC survey according to CLSI guidelines.

As a result, the antibacterial protein SBL2200 showed lytic activity only against streptococci and did not show lytic activity against other test strains. The results are presented in Table 2.

Antibacterial activity results fungus Strain Susceptibility MIC (μg/ml) Streptococcus agalactiae ATCC 27956 Susceptible 6.25 CCARM 4523 Susceptible 6.25 CCARM 4537 Susceptible 6.25 Streptococcus pyogenes CCARM 4528 Susceptible 1.56 CCARM 4533 Susceptible 1.56 Staphylococcus aureus ATCC 33591 Insusceptible - CCARM 3090 Insusceptible - Acinetobacter baumannii CCARM 12202 Insusceptible - Clostridioides difficile CCARM 0186 Insusceptible - Pseudomonas aeruginosa ATCC BAA-47 Insusceptible - Klebsiella pneumoniae CCARM 10332 Insusceptible - Escherichia coli CCARM 1G938 Insusceptible -

From these results, it was confirmed that the antibacterial activity of the antibacterial protein SBL2200 of the present invention is highly specific to streptococci.

From the above results, it was confirmed that the streptococcus-specific antibacterial protein SBL2200 of the present invention can ultimately kill streptococci by lysing them. These characteristics demonstrate that a pharmaceutical composition containing the streptococcus-specific antibacterial protein SBL2200 can be used for the purpose of killing streptococci during streptococcal infection, and can also be used in the same way as conventional antibiotics for the purpose of treating streptococcal infection. It shows that it can be done.

Example 3: Streptococcus specific antibacterial protein SBL2200's streptococcus Treatment effect on infection inspection

The therapeutic effect of the antibacterial protein SBL2200 on streptococcal infection was investigated through infection animal model testing. Specifically, 5-week-old ICR mice [specific pathogen-free (SPF) grade] weighing approximately 20 g were used as test animals. A total of 20 mice were separated into 2 groups (10 mice per group), and then Streptococcus per mouse was administered intravenously. Infection was induced by administering 1×10 ⁸ cfu of agalactiae CCARM 4503 (i.e., 1×10 ⁸ cfu/mouse). For animals in the treatment group, 0.2 ml of a pharmaceutical composition of the antibacterial protein SBL2200 was intravenously administered 30 minutes, 12 hours, and 24 hours after forced infection with the bacteria. For the animals in the control group, the same volume of only buffer (50 mM Potassium phosphate, 5% (w/v) Sorbitol, 0.1% (w/v) poloxamer 188, pH 7.5) was administered. Buffer administration was performed 30 minutes, 12 hours, and 24 hours after forceful infection with bacteria, in the same manner as the administration of the pharmaceutical composition of the antibacterial protein SBL2200. The number of deaths was investigated every day for 5 days after forced infection with the bacteria, and the observation of any unusual phenomena was also investigated twice a day, in the morning and afternoon.

As a result, a clear treatment effect was confirmed. The number of deaths was as shown in Table 3 below, and administration of the pharmaceutical composition of the antibacterial protein SBL2200 of the present invention provided a clear improvement in the survival rate of infected animals. In addition, while various specific reactions such as redness of the lid margin and decreased activity were observed in the control group, no such specific reactions were observed in the animals in the treatment group administered the pharmaceutical composition of the antibacterial protein SBL2200.

Streptococcal infection treatment effect army number of deaths Number of dead animals/Number of test animals Mortality rate (%) Days after forced infection with bacteria One 2 3 4 5 control group
(Control) 0 3 One One 0 5/10 50 church
(Treatment) 0 0 0 0 0 0/10 0

From the above results, it was confirmed that the antibacterial protein SBL2200 of the present invention is effective in treating streptococcal infection. These characteristics show that a pharmaceutical composition containing the antibacterial protein SBL2200 as an active ingredient can be used for the treatment of streptococcal infections.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention. Accordingly, the practical scope of the present invention will be defined by the appended claims and their equivalents.

KCTC KCTC15033BP 20220712

Claims (5)

SBL2200, an antibacterial protein with streptococci-specific antibacterial activity and an amino acid sequence represented by SEQ ID NO: 1. The antibacterial protein SBL2200 according to claim 1, wherein the antibacterial protein SBL2200 consists of 279 amino acids and has a molecular weight of 31.3 kDa. A pharmaceutical composition for treating streptococcal infection comprising the antibacterial protein SBL2200 of claim 1 as an active ingredient. The pharmaceutical composition for treating streptococcal infection according to claim 3, wherein the pharmaceutical composition is used as an antibiotic, disinfectant, disinfectant, or therapeutic agent. Escherichia production strain produced by transforming E. coli using a plasmid represented by the nucleic acid sequence of SEQ ID NO: 2 Method for producing the antibacterial protein SBL2200 of claim 1 using coli AB-22 (accession number KCTC15033BP).